This invention relates to energy transfer between fluorochrome labeled proteins to determine analyte concentration and, more particularly, to methods of determining cAMP concentration using labeled cAMP dependent protein kinase.
The function of many biological molecules is mediated through interactions with proteins and protein complexes. Many of these molecules are found in extremely small concentrations within a living cell. However, their concentration may transiently change in response to physiological stimuli. In many instances, it is important to determine the concentration of such molecules to assess the metabolic state of a cell, hormonal responses and certain diseases. Even if the level changes several fold, at such minute concentrations, such change is not detectable.
One such important molecule which mediates biological function through protein interaction is cyclic AMP (cAMP, adenosine 3', 5'-cyclic monophosphate). Cyclic AMP regulates intracellular reactions in all procaryotic and nucleated animal cells that have been studied to date. It functions as a ubiquitous intracellular mediator, or second messenger, of a variety of hormone induced effects, the first messenger being the extracellular hormone. Such hormone induced effects mediated by increased cAMP levels include, for example, triglyceride breakdown in a fat tissue induced by epinephrine, adrenocorticotropic hormone (ACTH), glucagon or thyroid-stimulating hormone (TSH); water resorption in the kidney by vasopressin; glycogen breakdown in the muscle and liver; increased heart rate by epinephrine and progesterone secretion by the ovary in response to luteinizing hormone. These differential responses to cAMP are induced by hormone-receptor interactions at the cell surface and result in the intracellular synthesis of cAMP. Thus, different target cells respond to increases in cAMP levels in different but characteristic way.
For cAMP to function as a second messenger, its intracellular concentration must be tightly controlled and able to change rapidly in response to hormone-receptor binding. Normally, cAMP levels are less than about 1 .mu.M and increase approximately five-fold upon hormonal stimulation. The increase in cAMP levels is due to synthesis by the enzyme adenylate cyclase from ATP. Following this transient increase, cAMP levels are rapidly returned to normal levels through the action of phosphodiesterases.
To effectively study cAMP dependent hormonal responses and their physiological mechanisms, it is necessary to accurately measure small concentrations of cAMP. More essentially, it is important to measure free cAMP concentrations, since in general, the biological activity of intracellular messengers is controlled by and correlates with the concentrations of free messenger, whereas total concentrations measured after tissue disruption include material bound to biologically irrelevant sites. Previous methods for measuring cAMP concentrations employed classical competitive binding assays such as radioimmunoassays, and required the manipulation and preparation of extracts from thousands or millions of cells. These methods resulted in poor spatial and temporal resolution and were incapable of distinguishing free or biologically active cAMP levels from total or sequestered cAMP. Moreover, these methods required the use of hazardous, perishable isotopes and were therefore expensive.
Additionally, second messengers other than cAMP such as cyclic GMP (cGMP), calcium and diacylglycerol as well as other organic molecules such as tumor-promoting phorbol esters are of great importance in signal transduction and cell physiology. Accurate determination of the biologically active concentrations of these molecules is also necessary to effectively study hormonal responses and physiological mechanisms. However, except for simple inorganic ions such as calcium, there is no indicator system such as to measure the biologically active concentrations of these molecules. Most of the same drawbacks that exist for determining free cAMP concentrations within the cell exist for these molecules as well.
Thus, there exists a need for a method to rapidly, efficiently and non-destructively measure free intracellular cAMP concentrations as well as other organic molecules in single living cells. Such an invention would be of critical importance to the understanding of hormonal regulation. The present invention satisfies this need and provides related advantages as well.